Michael Ovadia

Isolation and characterization of a metalloproteinase with weak hemorrhagic activity from the venom of the snake Bothrops asper (terciopelo)

A metalloproteinase, named BaP1, was purified to homogeneity from the venom of Bothrops asper (Pacific region) of Costa Rica by ion-exchange chromatography on CM-Sephadex and gel filtration on Sephacryl S-200. The enzyme has a mol. wt of 24,000 and contains few Cys and high numbers of Asp, Leu, Ser and Glu. BaP1 hydrolyzes casein, hide powder azure and fibrinogen, having an optimal pH of 8.0. It rapidly digests the A alpha-chain of fibrinogen and, later on, the B beta-chain, leaving the gamma-chain unaffected. Chelating agents (EDTA and 1,10-phenanthroline) inhibited proteolytic activity, whereas 2-mercaptoethanol and soybean trypsin inhibitor did not affect this activity. BaP1 has a weak hemorrhagic activity, with a minimum hemorrhagic dose of 20 micrograms; this activity was inhibited by EDTA and was abolished after incubation at 60 degrees C. In addition, BaP1 induces edema and a mild myotoxic effect, lacking coagulant, defibrinating and lethal effects.

Orally administrated cinnamon extract reduces β-amyloid oligomerization and corrects cognitive impairment in Alzheimer's disease animal models.

An increasing body of evidence indicates that accumulation of soluble oligomeric assemblies of β-amyloid polypeptide (Aβ) play a key role in Alzheimers disease (AD) pathology. Specifically, 56 kDa oligomeric species were shown to be correlated with impaired cognitive function in AD model mice. Several reports have documented the inhibition of Aβ plaque formation by compounds from natural sources. Yet, evidence for the ability of common edible elements to modulate Aβ oligomerization remains an unmet challenge. Here we identify a natural substance, based on cinnamon extract (CEppt), which markedly inhibits the formation of toxic Aβ oligomers and prevents the toxicity of Aβ on neuronal PC12 cells. When administered to an AD fly model, CEppt rectified their reduced longevity, fully recovered their locomotion defects and totally abolished tetrameric species of Aβ in their brain. Furthermore, oral administration of CEppt to an aggressive AD transgenic mice model led to marked decrease in 56 kDa Aβ oligomers, reduction of plaques and improvement in cognitive behavior. Our results present a novel prophylactic approach for inhibition of toxic oligomeric Aβ species formation in AD through the utilization of a compound that is currently in use in human diet.

Isolation and characterization of synergistic hemorrhagins from the venom of the snake Bothrops asper

Three hemorrhagic factors (BaH1, BH2 and BH3) were isolated from the venom of Bothrops asper by gel filtration on Sephacryl S-200, DEAE-Sepharose chromatography, metal chelate affinity chromatography and hydrophobic interaction chromatography. They contain 55% of the total hemorrhagic activity of the whole venom when they are mixed, but lose almost half of the activity if they are separated, indicating a synergism between the three. The main hemorrhagin is BaH1 (Bothrops asper hemorrhagin 1); the other two are weak hemorrhagins but contribute to the synergism. They are acidic proteins with a pI of 4.5, 5.2 and 5; their mol. wt is 64,000, 26,000 and 55,000 respectively. The minimal hemorrhagic dose (MHD) of BaH1, BH2 and BH3 is 0.18, 2 and 1.6 micrograms, with a specific activity 55, 5 and 6.25 higher than that of the whole venom. The hemorrhagic activity of all three factors was inhibited by EDTA and ortho-phenathroline, indicating that the hemorrhagic activity is metal dependent. Phosphoramidon, soybean trypsin inhibitor, PMSF, pepstatin and aprotinin did not affect the hemorrhagic activity of the isolated factors.

Neutralization of Viperidae and Elapidae snake venoms by sera of different animals

The venoms of three vipers, Vipera palaestinae, Echis colorata and Pseudocerastes fieldi, and the elapid Walterinnesia aegyptia, all from Israel, show a similar ld50 of 250–300 μg/kg body weight when injected intravenously into mice. The venoms of the viperid Aspis cerastes from Israel and the elapid Naja nigricollis from East Africa are less toxic and have an ld50 of 600 and 1200 μg/kg respectively. Compared with mice on a weight basis, V. palaestinae, Natrix tessellata (a non-venomous water snake) and the mammal Herpestes ichneumon (mongoose) are highly resistant to V. palaestinae venom; Mesocricetus auratus (hamster) also shows some degree of tolerance. The sera from these and other animals were tested for their capacity to neutralize this venom in vitro. The snake sera, which represented the families Elapidae, Viperidae, Crotalidae and Colubridae, all neutralized V. palaestinae venom, whereas sera from two lizards (Uromastix aegyptius and Agama stellio) failed to do so. Of the mammalian sera tested, neutralization was clearly demonstrated with hamster serum and to a lesser degree with hedgehog (Erinaceus europeus) serum, but not with human serum, cat serum or rabbit serum, and even serum from the highly resistant mongoose failed to show any neutralization. In tests involving these sera and the 6 venoms listed above, the Viperidae venoms were neutralized by sera from snakes of the same family, by W. aegyptia serum, and, with the exception of E. colorata venom, by N. nigricollis serum. The neutralization by sera from non-venomous snakes was slightly less complete, and sera from the mongoose and hamster failed to neutralize in some cases. N. nigricollis venom could be neutralized by the homologous serum and by W. aegyptia serum, but not by any others. All test sera, including the homologous serum, were without effect on W. aegyptia venom. Despite the lack of neutralizing capacity of its serum, W. aegyptia is highly resistant to its own venom and the mongoose to both of the elapid venoms tested. This resistance is apparently not due to humoral factors.

Pathological changes induced by BaH1, a hemorrhagic proteinase isolated from Bothrops asper (Terciopelo) snake venom, on mouse capillary blood vessels.

The pathological changes induced in capillaries by BaH1, a hemorrhagic metalloproteinase isolated from the venom of Bothrops asper, were studied after i.m. injection in mouse gastrocnemius. Hemorrhage was observed macroscopically, and corroborated histologically, within the first 5 min. At the ultrastructural level, the earliest changes in endothelial cells, observed 1 min after toxin administration, consisted of a decrease in the number of pinocytotic vesicles, the presence of blebs and cytoplasmic projections pinching off to the vascular lumen and the detachment of endothelial cells from the surrounding basal lamina. These processes occurred concomitantly with a thinning of endothelial cells. In capillaries undergoing more advanced degenerative stages, there were gaps or breaks in endothelial cells through which erythrocytes were escaping to the extravascular space. In these cells, the basal lamina was usually absent. Throughout this process, intercellular junctions remained apparently intact and no evidence was found of extravasation through widened intercellular junctions. In addition to this morphological pattern of degeneration, some capillaries presented swollen endothelial cells with dilated endoplasmic reticulum and lacking pinocytotic vesicles. Many capillaries contained platelet plugs and fibrin. Thus, hemorrhage induced by BaH1 occurs per rhexis, as has been also described for other venoms and hemorrhagic toxins.

Purification and characterization of an antihemorrhagic factor from the serum of the snake Vipera palaestinae

Abstract The serum of Vipera palaestinae contains a factor which neutralizes effectively all the hemorrhagic activities of its venom. Antibodies prepared in rabbits against the crude snake serum do not abolish its antihemorrhagic activity. The antihemorrhagic factor was purified from the snake serum by ammonium sulfate precipitation, Sephadex G-75 gel filtration, DEAE-cellulose chromatography and isoelectrofocusing. The purified protein shows one band in immunoelectrophoresis and in disc electrophoresis which strongly stains with the PAS procedure. It has no gelatinase or caseinase activity; it is digestible by trypsin and is stable at high temperatures and at pHs of 6·0–9·5. The neutralization capacity of the purified preparation is about 20 times that of the specific horse immunoglobulins. The molecular weight of the antihemorrhagic factor is about 80,000 and its isoelectric point is 4·7. These data, together with the failure to form precipitin lines in immunodiffusion tests, suggest that the antihemorrhagic factor of Vipera palaestinae serum is probably an albumin-like or α-globulin fraction rather than an immunoglobulin. The antihemorrhagic factor of V. palaestinae also neutralizes the hemorrhagic activity of the viperids Echis colorata and Cerastes cerastes .

Isolation and characterization of three hemorrhagic factors from the venom of Vipera palaestinae

Abstract Three hemorrhagic factors were purified from the venom of Vipera palaestinae by gel filtration on Sephadex G-75, ammonium sulfate precipitation and DEAE-cellulose chromatography. One hemorrhagic factor is basic (HR1), one is weakly acidic and one is strongly acidic. All these factors have a similar mol. wt of about 60,000 and each of them shows one precipitin line in immunoelectrophoresis. HR1 and HR2 show both gelatinase and caseinase activity; HR3 has no detectable proteolytic activity. The basic hemorrhagic principle is a glycoprotein (positive PAS staining). Together with the weekly acidic factor it accounts for most of the hemorrhagic activity; the specific activity of each of these two components is 10 times higher than that of the crude venom.

Activity of hemorrhagic metalloproteinase BaH-1 and myotoxin II from Bothrops asper snake venom on capillary endothelial cells in vitro

In vivo, hemorrhagic toxins isolated from snake venoms cause a disorganization of the basal lamina of capillaries, with a concomitant degenerative process of endothelial cells. In this study we investigated the effects of BaH-1, a hemorrhagic metalloproteinase purified from the venom of Bothrops asper, on a murine endothelial cell line of capillary origin. A quantitative cytotoxicity assay based on the release of lactic dehydrogenase was utilized. BaH-1, despite its potent hemorrhagic activity, did not exert direct cytolytic activity on the endothelial cells, even at concentrations as high as 65 micrograms/ml. The only visible effect of BaH-1 on the cultured cells was a relatively slow, moderate detachment of cells, interpreted as a consequence of proteolytic degradation of extracellular matrix components. In contrast, myotoxin II, a lysine-49 phospholipase A2 from the same venom, was clearly cytotoxic to this cell type, albeit being devoid of hemorrhagic activity. These findings suggest that the ability of venom metalloproteinases to induce hemorrhage is not related to a direct cytotoxic action on endothelial cells, and that the rapid degenerative changes of endothelium observed in vivo are probably the result of an indirect mechanism.

Ultrastructural alterations in mouse capillary blood vessels after experimental injection of venom from the snake Bothrops asper (Terciopelo)

Histological and ultrastructural alterations in capillary blood vessels were studied at various time intervals after im injection of 50 micrograms of Bothrops asper snake venom in mouse gastrocnemius muscle. Hemorrhage was observed as early as 5 min after envenomation, as abundant erythrocytes appeared in the interstitial space. Ultrastructural observations revealed two different patterns of pathological changes: in the majority of damaged capillaries, endothelial cells had blebs and cytoplasmic projections pinching off to the lumen. This phenomenon was observed together with a decrease in the number of pinocytotic vesicles, with endothelial cells becoming very thin. As an apparent consequence of this process, some endothelial cells had evident gaps in their continuity. In addition, basal laminae surrounding these capillaries were altered and discontinuous. Other endothelial cells underwent a morphologically different process of degeneration, characterized by swelling of the endoplasmic reticulum and of the cytosol. These cells had a diffuse appearance and their basal laminae were discontinuous or absent. No major changes in the intercellular junctions were noticed in damaged endothelial cells. Samples obtained 30 and 60 min after venom injection were devoid of normal capillaries in many areas, and only diffuse remnants of their structure were found. Many altered capillaries had platelet aggregates and fibrin, the latter also being observed in the interstitial space. It is concluded that B. asper venom induces rapid and drastic pathological effects on capillaries leading to hemorrhage per rhexis i.e., erythrocytes probably escape through gaps in damaged endothelial cells and not through widened intercellular junctions.

Localization of the Vitellin and Its Possible Precursors in Various Organs of Parapenaeus longirostris (Crustacea, Decapoda, Penaeidae)

Summary Histological studies and immunofluorescence revealed two phases of cytoplasmic development during oogenesis of Parapenaeus longirostris. The first phase includes synthesis of a substance which accumulates later in the peripheral cortical granules. During the second phase, yolk globules appear in the cytoplasm. Immunodiffusion, Immunoelectrophoresis and immunofluorescence demonstrated materials immunologically similar to vitellin in the yolk globules of developed oocytes but not in oocytes in the first phase of development. This material appears also in the subepidermal adipose tissue and in the hemolymph, indicating the extraovarian origin of vitellin.